Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
권호 표지

Methylation of the Mouse Dlx5 and Osx Gene Promoters Regulates Cell Type-specific Gene Expression

  • Lee, Ji Yun (Department of Anatomy, School of Medicine, Kyungpook National University) ;
  • Lee, Yu Mi (Department of Anatomy, School of Medicine, Kyungpook National University) ;
  • Kim, Mi Jin (Department of Anatomy, School of Medicine, Kyungpook National University) ;
  • Choi, Je Yong (Skeletal Diseases Genome Research Center, Kyungpook National University Hospital) ;
  • Park, Eui Kyun (Skeletal Diseases Genome Research Center, Kyungpook National University Hospital) ;
  • Kim, Shin Yoon (Skeletal Diseases Genome Research Center, Kyungpook National University Hospital) ;
  • Lee, Sam Poong (Department of Genetic Engineering, Daegu University) ;
  • Yang, Jae Sup (Department of Genetic Engineering, Daegu University) ;
  • Kim, Dong Sun (Department of Anatomy, School of Medicine, Kyungpook National University)
  • Received : 2006.05.11
  • Accepted : 2006.08.08
  • Published : 2006.10.31
⟨ Previous Next ⟩

Abstract

Dlx5 and Osx are master regulatory proteins essential for initiating the cascade leading to osteoblast differentiation in mammals, but the mechanism of osteoblast-specific expression is not fully understood. DNA methylation at CpG sequences is involved in tissue and cell type-specific gene expression. We investigated the methylation status of Dlx5 and Osx in osteogenic and nonosteogenic cell lines by methylationspecific PCR (MSP). The CpG dinucleotides of the Dlx5 and Osx promoter regions were unmethylated in osteogenic cell lines transcribing these genes but methylated in nonosteogenic cell lines. Treatment of C2C12 cells with 5-AzadC induced dose- and timedependent expression of Dlx5 and Osx mRNA by demethylating the corresponding promoters. Furthermore the mRNAs for the osteoblast markers ALP and OC, which were undetectable in untreated cells, gradually increased after 5-AzadC treatment. In addition, BMP-2 stimulation induced Dlx5 expression by hypomethylating its promoter. These findings suggest that DNA methylation plays an important role in cell type-specific expression of Dlx5 and Osx.

Keywords

Acknowledgement

Supported by : Korea Research Foundation

References

  1. Acampora, D., Merio, G. R., Paleari, L., Zerega, B., Postiglione, M. P., et al. (1999) Craniofacial, vestibular and bone defects in mice lacking the Dist-less-related gene Dlx5. Development 126, 3795-3809
  2. Attwood, J. T., Yung, R. L., and Richardson, B. C. (2002) DNA methylation and the regulation of gene transcription. Cell. Mol. Life Sci. 59, 241-257 https://doi.org/10.1007/s00018-002-8420-z
  3. Beanan, M. J. and Sargent, T. D. (2000) Regulation and function of Dlx3 in vertebrate development. Dev. Dyn. 218, 545-553 https://doi.org/10.1002/1097-0177(2000)9999:9999<::AID-DVDY1026>3.0.CO;2-B
  4. Bird, A. P. and Wolffe, A. P. (1999) Methylation-induced repression- belts, braces, and chromatin. Cell 99, 451-454 https://doi.org/10.1016/S0092-8674(00)81532-9
  5. Budden, S. S. and Gunness, M. E. (2003) Possible mechanisms of osteopenia in Rett syndrome: Bone histomorphometric studies. J. Child Neurol. 18, 698-702 https://doi.org/10.1177/08830738030180100401
  6. Eden, S., Hashimshony, T., Keshet, I., Cedar, H., and Thorne, A. W. (1998) DNA methylation models histone acetylation. Nature 394, 842 https://doi.org/10.1038/29680
  7. Ehrlich, M. (2003) Expression of various genes is controlled by DNA methylation during mammalian development. J. Cell. Biochem. 88, 899-910 https://doi.org/10.1002/jcb.10464
  8. Gidekel, S. and Bergman, Y. (2002) A unique developmental pattern of Oct-3/4 DNA methylation is controlled by a cisdemodification element. J. Biol. Chem. 277, 34521-34530 https://doi.org/10.1074/jbc.M203338200
  9. Gruenbaum, Y., Stein, R., Cedar, H., and Razin, A. (1981) Methylation of CpG sequences in eukaryotic DNA. FEBS Lett. 124, 67-71 https://doi.org/10.1016/0014-5793(81)80055-5
  10. Harada, S. and Rodan, G. A. (2003) Control of osteoblast function and regulation of bone mass. Nature 423, 349-355 https://doi.org/10.1038/nature01660
  11. Hass, R. H., Dixon, S. D., Sartoris, D., and Hennessy, M. J. (1997) Osteopenia in Rett syndrome. J. Pediatr. 13, 771-774
  12. Hassan, M. Q., Javed, A., Morasso, M. I., Karlin, J., Montecino, M., et al. (2004) Dlx3 transcriptional regulation of osteoblast differentiation: temporal recruitment of Msx2, Dlx3, and Dlx5 homeodomain proteins to chromatin of the osteocalcin. Mol. Cell. Biol. 24, 9248-9261 https://doi.org/10.1128/MCB.24.20.9248-9261.2004
  13. Herman, J. G., Graff, J. R., Myohanen, S., Nelkein, B. D., and Baylin, S. B. (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl. Acad. Sci. USA 93, 9821-9826
  14. Hershko, A. Y., Kafri, T., Fainsod, A., and Razin, A. (2003) Methylation of HoxA5 and HoxB5 and its relevance to expression during mouse development. Gene 302, 65-72 https://doi.org/10.1016/S0378111902010910
  15. Imamura, T., Ohgane, J., and Ito, S. (2001) CpG island of rat sphingosine kinase-1 gene: tissue-dependent DNA methylation status and multiple alternative first exons. Genomics 76, 117-125 https://doi.org/10.1006/geno.2001.6607
  16. Jones, P. A. and Taylor, S. M. (1980) Cellular differentiation, cytidine analogs and DNA methylation. Cell 20, 85-93 https://doi.org/10.1016/0092-8674(80)90237-8
  17. Juttermann, R., Li, E., and Jaenisch, R. (1994) Toxicity of 5′- aza-2′-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation. Proc. Natl. Acad. Sci. USA 91, 11797-11801
  18. Katagiri, T. and Takashima, N. (2002) Regulatory mechanisms of osteoblast and osteoclast differentiation. Oral Dis. 8, 147- 159 https://doi.org/10.1034/j.1601-0825.2002.01829.x
  19. Kim, J. H., Hwang, E. H., Park, H. J., Paik, Y. K., and Shim, Y. H. (2005) Methylation of CpG islands in the rat 7-dehydrocholestrol reductase promoter suppresses transcriptional activation. Mol. Cells 19, 279-282 https://doi.org/10.1016/j.molcel.2005.05.028
  20. Kitazawa, S. and Kitazawa, R. (2002) Epigenetic control of mouse receptor activator of NF-$\kappa$B ligand gene expression. Biochem. Biophys. Res. Commun. 293, 126-131 https://doi.org/10.1016/S0006-291X(02)00189-4
  21. Lander, E. S., Linton, L. M., Birren, B., Nusbaum, C., Zody, M. C., et al. (2001) Initial sequencing and analysis of the human genome. Nature 409, 860-921 https://doi.org/10.1038/35057062
  22. Lee, M. H., Kim, Y. J., Kim, H. J., Park, H. D., Kang, A. R., et al. (2003) BMP-2-induced Runx2 expression is mediated by Dlx5 and TGF-1 opposes the BMP-2-induced osteoblast differentiation by suppression of Dlx5 expression. J. Biol. Chem. 278, 34387-34394 https://doi.org/10.1074/jbc.M211386200
  23. Leonard, H., Thomson, M. R., and Glasson, E. J. (1999) A population based approach to the investigation of osteopenia in Rett syndrome. Dev. Med. Child Neurol. 41, 323-328 https://doi.org/10.1017/S0012162299000717
  24. Li, E. (2003) Chromatin modification and epigenetic reprogramming in mammalian development. Nat. Genet. 3, 662- 673
  25. Locklin, R. M., Oreffo, R. O. C., and Trifitt, J. T. (1998) Modulation of osteogenic differentiation in human skeletal cells in vitro by 5-azacytidine. Cell Biol. Int. 22, 207-215 https://doi.org/10.1006/cbir.1998.0240
  26. Maatouk, D. M. and Resnick, J. L. (2003) Continuing primordial germ cell differentiation in the mouse embryo is a cellintrinsic program sensitive to DNA methylation. Dev. Biol. 258, 201-208 https://doi.org/10.1016/S0012-1606(03)00110-6
  27. Nakashima, K., Zhou, X., Kunkel, G., Zhang, Z., Deng, J. M., et al. (2002) The novel zinc finger-containing transcription factor Osterix is required for osteoblast differentiation and bone formation. Cell 108, 17-29 https://doi.org/10.1016/S0092-8674(01)00622-5
  28. Nishino, K., Hattori, N., Tanaka, S., and Shiota, K. (2004) DNA methylation-mediated control of Sry gene expression in mouse gonadal development. J. Biol. Chem. 279, 22306- 22313 https://doi.org/10.1074/jbc.M309513200
  29. Park, G. T. and Morasso, M. I. (1999) Regulation of the Dlx3 homeobox gene upon differentiation of mouse keratinocytes. J. Biol. Chem. 274, 26599-26608 https://doi.org/10.1074/jbc.274.37.26599
  30. Plachot, C. and Lelievre S. A. (2004) DNA methylation control of tissue polarity and cellular differentiation in the mammary epithelium. Exp. Cell Res. 298, 122-132 https://doi.org/10.1016/j.yexcr.2004.04.024
  31. Raman, R. and Narayan, G. (1995) 5-Aza-2′-deoxycytidineinduced inhibition of differentiation of spermatogonia to spermatocytes in the mouse. Mol. Reprod. Dev. 42, 284-290 https://doi.org/10.1002/mrd.1080420304
  32. Razin, A., Webb, C., Szyf, M., Yisraeli, J., Rosenthal, A., et al. (1984) Variations in DNA methylation during mouse cell differentiation in vivo and in vitro. Proc. Natl. Acad. Sci. USA 81, 2275-2279
  33. Ryhanen, S., Pirskanen, A., Jaaskelainen, T., and Maenpaa, P. H. (1997) State of methylation of the human osteocalcin gene in bone-derived and other types of cells. J. Cell. Biochem. 66, 404-412 https://doi.org/10.1002/(SICI)1097-4644(19970901)66:3<404::AID-JCB12>3.0.CO;2-E
  34. Ryoo, H. M., Hoffmann, H. M., Beumer, T., Frenkel, B., Towler, D. A., et al. (1997) Stage-specific expression of Dlx-5 during osteoblast differentiation: involvement in regulation osteocalcin gene expression. Mol. Endocrinol. 11, 1681-1694 https://doi.org/10.1210/me.11.11.1681
  35. Sengupta, P. K. and Smith, B. D. (1998) Methylation in the initiation region of the first exon suppress collagen pro-$\alpha$ 2(I) gene transcription. Biochim. Biophys. Acta 1443, 75-89 https://doi.org/10.1016/S0167-4781(98)00188-2
  36. Shiota, K., Kogo, Y., Ohgane, J., Imamura, T., Urano, A., et al. (2002) Epigenetic marks by DNA methylation specific to stem, germ and somatic cells in mice. Genes Cells 7, 961-969 https://doi.org/10.1046/j.1365-2443.2002.00574.x
  37. Suzuki, Y., Tsunoda, T., Sese, J., Taira, H., Mizushima-Suguno, J., et al. (2001) Identification and characterization of the potential promoter regions of 1031 kinds of human genes. Genome Res. 11, 677-684 https://doi.org/10.1101/gr.GR-1640R
  38. Takizawa, T., Nakashima, K., Namihira, M., Ochiai, W., Uemura, A., et al. (2001) DNA methylation is a critical cellintrinsic determinant of astrocyte differentiation in the fetal brain. Dev. Cell 1, 749-758 https://doi.org/10.1016/S1534-5807(01)00101-0
  39. Taylor, S. M. and Jones, P. A. (1979) Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine. Cell 17, 771-779 https://doi.org/10.1016/0092-8674(79)90317-9
  40. Uehara, R., Suzuki, Y., and Ichikawa, Y. (2001) Methotrexate (MTX) inhibits osteoblastic differentiation in vitro: Possible mechanism of MTX osteopathy. J. Rheumat. 28, 251-256
  41. Villagra, A., Gutierrez, J., Paredes, R., Sierra, J., Puchi, M., et al. (2002) Reduced CpG methylation is associated with transcriptional activation of the bone-specific rat osteocalcin gene in osteoblast. J. Cell. Biochem. 85, 112-122 https://doi.org/10.1002/jcb.10113
  42. Zuscik, M. J., Baden, J. F., Wu, Q., Sheu, T. J., Schwarz, E. M., et al. (2004) 5-Azacytidine alters TGF-$\beta$ and BMP signaling and induces maturation in articular chondrocytes. J. Cell. Biochem. 92, 316-331 https://doi.org/10.1002/jcb.20050